Anticancer composition comprising obovatol or obovatal

ABSTRACT

Disclosed herein is an anticancer composition, comprising obovatol, represented by the following Chemical Formula 1, obovatal, represented by the following Chemical Formula 2, and/or pharmaceutical salts thereof. The composition exhibits the activity of inhibiting the growth of cancer cells and suppressing the expression and activity of matrix metalloproteinases (MMPs), and thus can be useful for the prophylaxis and treatment of cancer as well as for the inhibition of cancer metastasis.

This Application claims priority to Republic of Korea patent applicationserial number 10-2005-0073915, filed Aug. 11, 2005, which is hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to an anticancer composition containingobovatol or obovatal as an active ingredient.

BACKGROUND ART

With the great progress of civilization, cancer morbidity has beenreported to increase. Despite the increasing incidence of cancer,therapies for cancer still fall within the scope of surgical operation,radiotherapy, and chemical therapies using about 40 chemicals havingstrong cytotoxicity. However, these therapies are useful only in cancerpatients in early stages, or are effective only for certain types ofcancer. Despite these therapies, cancer mortality is currently tendingto increase.

Death from cancer is mainly due to the metastasis of cancer cells intoother tissues rather than due to initial tumors. Active research on theinfiltrative and metastatic mechanism of cancer cells and the preventionthereof has been conducted in an effort to reduce cancer mortality.

To metastasize, a cancer cell must break away from the primary tumor,invade support structures of normal tissue, such as interstitial spaceor capillary basement membranes, to attach to either the circulatory orlymph system, degrade extracellular matrixes and basement membranes,circulate through the bloodstream, and grow at distant loci(metastasize) as a secondary tumor in normal tissues elsewhere in thebody, with the concurrent generation of blood vessels (angiogenesis).

Accordingly, the process of metastasis is mainly comprised ofattachment, invasion and angiogenesis. If any of them is prevented fromhappening, cancer metastasis can be prevented. In the invasion of cancercells, matrix metalloproteinases (MMPs), secreted from the attachedcells, are known to play important roles.

MMPs are enzymes involved in the degradation of the extracellular matrix(ECM), such as collagen, proteoglycans, etc, and are represented by, forexample, interstitial collagenase, MMP-2, and stromelysin. Also, MMPs, afamily of zinc-dependent endopeptidases, must undergo zymogen activationby other proteases or organic phosphorus compounds prior to expressingany proteolytic activity. Their activity is inhibited by TIMP (tissueinhibitor of metalloproteinase) that is secreted together with MMPs.Sharing high cDNA sequence homology, MMPs are collectively classified asa family. MMPs are found to be involved in many pathological conditions,including abnormal connective tissues and basement membrane matrixmetabolism, such as tissue ulceration, abnormal wound healing,periodontal disease, bone disease, tumor metastasis or invasion as wellas HIV infection (J. Leuk. Biol., 52 (2): 244-248, 1992). In fact,cancer cells, which actively metastasize, show high activity of MMP2 orMMP9, compared to normal cells or non-metastatic cancer cells, andinhibitors of the MMPs are shown to prevent cancer metastasis.

With this background, the present inventors succeeded in isolating andpurifying obovatol and obovatal from Magnoliaceae (Magnolia obovataThunberg), which has been used as a herbal medicine, and found that suchcompounds have the activity of inhibiting the expression and enzymaticactivity of MMPs, which play pivotal roles in the growth and metastasisof various human cancer cells.

DISCLOSURE OF THE INVENTION

Accordingly, the present invention has been made keeping in mind theabove problems occurring in the prior art, and an object of the presentinvention is to provide an anticancer composition containing obovatol,represented by the following Chemical Formula 1, and/or obovatal,represented by the following Chemical Formula 2, as a therapeuticallyactive ingredient.

Another object of the present invention is to provide a method for theprophylaxis, treatment and/or metastatic inhibition of cancers using ananticancer composition containing obovatol, represented by the followingChemical Formula 1, and/or obovatal, represented by the followingChemical Formula 2, as a therapeutically active ingredient.

A further object of the present invention is to provide a method forisolating and purifying obovatol and obovatal, represented by thefollowing Chemical Formulas 1 and 2, respectively, from magnoliacea.

BEST MODE FOR CARRYING OUT THE INVENTION

In one embodiment, the present invention provides an anticancercomposition containing obovatol, represented by the following ChemicalFormula 1, obovatal, represented by the following Chemical Formula 2, orpharmaceutically acceptable salts thereof.

Obovatol and/or obovatal, as an active ingredient of the composition, isfound to effectively inhibit the expression and enzymatic activity ofMMPs as well as the growth of cancer cells, in accordance with thepresent invention. Also, the administration of obovatol or obovatal via,for example, the oral route results in the inhibition of canceroustissues as identified in immune deficient mice into which rectal cancercells have been transplanted. With the anticancer effect of obovatol orobovatal, which is previously mentioned nowhere, the composition of thepresent invention is useful for the prophylaxis and treatment of canceror the inhibition of cancer matastasis.

The term “anticancer”, as used herein, is intended to refer to theactivity of suppressing the formation or growth of cancer cells, killingcancer cells, or inhibiting or blocking the metastasis of cancer cells,encompassing the meaning of the inhibition of cancer cell metastasis aswell as the prophylaxis and treatment of cancer.

The term “prophylaxis” as used herein is intended to mean all actionsintended to suppress the formation of cancer or retard cancer incidenceby administering the composition. In the present invention, the term“treatment” is intended to mean all actions intended to improve orbeneficially modify symptoms of the diseases by administering thecomposition.

The composition of the present invention can be preferably applied tolarge intestine cancer, stomach cancer, prostate cancer, breast cancer,kidney cancer, liver cancer, encephaloma, lung cancer, uterine cancer,colon cancer, bladder cancer, pancreatic cancer, and blood cancer forthe prophylaxis, treatment or metastatic inhibition thereof, but are notlimited thereto.

For use in the composition of the present invention, the compounds ofChemical Formulas 1 and 2 may be in the form of their salts, includingvarious pharmaceutically or physiologically acceptable organic orinorganic acid addition salts. Suitable as inorganic acids for formingsalts are hydrochloric acid, sulfuric acid and phosphoric acid. Examplesof suitable organic acids include carboxylic acid, phosphonic acid,sulfonic acid, acetic acid, propionic acid, octanoic acid, decanoicacid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipicacid, malic acid, tartaric acid, citric acid, glutamic acid, asparticacid, maleic acid, benzoic acid, salicylic acid, phthalic acid,phenylacetic acid, benzene sulfonic acid, 2-naphthalenesulfonic acid,methyl sulfuric acid, ethyl sulfuric acid, and dodecyl sulfuric acid.

In the composition of the present invention, the compounds of ChemicalFormula 1 and 2 or their salts may be used alone or in combination withone another and optionally in combination with pharmaceutically andphysiologically acceptable additives such as carriers, expedients,diluents, etc.

According to the intended administration and usage, the composition ofthe present invention may be prepared into enteral formulations, such aspowders, granules, tablets, capsules, suspensions, emulsions, syrup,aerosol, etc., or parenteral formulations, such as sterile injectionsolutions, and may be administered orally or injected via variousroutes, such as intraveneous, intraperitoneal, subcutaneous, rectal,local, etc., routes. Examples of carriers, expedients or diluentssuitable for use in the composition include lactose, dextrose, sucrose,sorbitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate,gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, non-crystalline cellulose, polyvinyl pyrrolidone, water,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate,and mineral oils. Optionally, the composition may further comprisefillers, anti-aggregates, lubricants, wetting agents, flavors,emulsifying agents, preservatives, etc.

Solid formulations suitable for oral administration include tablets,pills, powders, granules, capsules, etc. and may be formulated with atleast one expedient, such as calcium carbonate, sucrose, lactose,gelatin, etc. In addition to these, a lubricant, such as magnesiumstearate, talc, etc., may be contained in the composition of the presentinvention.

Liquid formulations suitable for oral administration, exemplified bysuspensions, peroral solutions, emulsions, syrup, etc., may comprisevarious expedients, such as wetting agents, sweetening agents, flavors,preservatives, as well as simple diluents such as water, liquidparaffin, etc.

As for non-oral formulations, sterile aqueous solutions, non-aqueoussolutions, suspensions, emulsions, lyophilized injections,suppositories, etc. may be exemplary. Non-aqueous solutions andsuspensions may contain propylene glycol, polyethylene glycol, vegetableoil such as olive oil, and injectable esters such as ethylolate.

In another embodiment, the present invention provides a method for theprophylaxis, treatment and/or metastatic inhibition of cancer by theadministration of obovatol of Chemical Formula 1, obovatal of ChemicalFormula 2, and/or pharmaceutically acceptable salts thereof to patients.

As used herein, the term “administration” is intended to refer to theprovision of the substance of interest in a suitable manner into thebodies of the patients. Administration routes of the composition of thepresent invention are not limited if they lead the active ingredient ofthe composition to the tissue of target, for example, orally orparenterally. In addition, the composition of the present invention maybe administered with the aid of a device for guiding the activeingredient to target cells.

The term “patients”, as used herein, is intended to mean subjects whosesymptoms are to be improved when administered with the composition ofthe present invention, including humans and animals, such asanthropoids, dogs, goats, pigs, rats, mice and the like. Accordingly,the composition of the present invention can be provided not only forhumans, but is also applicable to animals (for the purpose of theprophylaxis, treatment, and metastatic inhibition of cancer).

The composition of the present invention is administered in apharmaceutically effective amount. The term “pharmaceutically effective”means sufficient to treat diseases at reasonable ratios of beneficenceto danger for medicinal therapy. Dosages of the compound of the presentinvention depend on the kind and severity of diseases, activity of thedrug, sensitivity to the drug, frequency and time period ofadministration, administration routes, excretion rates, and factors wellknown in the art including, for example, concurrently used drugs, etc.The composition of the present invention may be used as a singlemedicine or in combination with other medicines concurrently orsequentially, and may be administered in single dosages or multipledosages. Taking into account the elements mentioned above, it isimportant to determine the dosage that elicits maximum therapeuticeffects without undesirable side effects, which is easy for thoseskilled in the art.

Depending on patients' age, sex, and weight, the compound according tothe present invention may be administered in a total dosage from 1 to 50mg per kg of weight, and preferably in a dosage from 1 to 10 mg per kgof weight, once or three times a day. Administration may be conductedevery day or every other day. However, because dosages may vary with theadministration route, disease severity, gender, weight, age, etc., itmust be understood that the above-mentioned dosage does not limit thescope of the present invention.

Obovatol, of Chemical Formula 1, or obovatal, of Chemical Formula 2, maybe prepared by being isolated from natural sources or by beingsynthesized using well-known methods. As a natural source for thecompound of Chemical Formula 1 or 2, silver magnolia is preferably used.

Silver magnolia [Magnolia obovata Thunberg], belonging to a Magnoliaceaefamily, is a deciduous tree about 5 m tall, which grows naturally inKorea, Japan, and China.

In herbal medicine, dried cortexes from silver magnolia have been usedfor the treatment of stomach diseases. Much has been reported about thevarious physiological activities of honokiol and magnolol, which can beextracted from leaves and cortexes of magnolia, but nowhere has theanticancer effect of obovatol and obovatal from magnolia been mentionedin previous literature.

In a further embodiment, the present invention is concerned with amethod for purifying obovatol and obovatal, comprising the steps of (1)extracting obovatol and obovatal from magnolia using alcohol; (2)subjecting the extract of step (1) to silica gel chromatography to yieldeluates; (3) subjecting the eluates of step (2) to thin layerchromatography; and (4) subjecting fractions of step (3) to highperformance liquid chromatography.

In the alcohol extraction step, leaves, fruit, bark, or all of them maybe used. For use in extraction, the alcohol is not specifically limited,but is preferably methanol.

In an example, the extract from leaves, bark and fruit in methanol isloaded on a silica gel chromatography column and eluted with ethylacetate and hexane to obtain active fractions. Next, thin layerchromatography is conducted by adsorbing the active fractions onto a C18chromatography column and eluting with methanol and water. Highperformance liquid chromatography using a methanol:water gradient ofpreferably 50:50 to 70:30 as an eluent results in the purification ofthe compound with high purity.

The purified compound was found to be obovatol or obovatal, as measuredby UV absorbance, IR absorbance, high resolution mass analysis, and NMRanalysis.

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

EXAMPLE 1 Extraction and Purification of Obovatol and Obovatal fromMagnolia

Magnolia leaves, fruit, or bark (taken from trees naturally growing inthe central region of Korea) were cut into pieces, and allowed to standfor 48 hours at room temperature while being immersed in methanol. Usingfilter paper, solids were filtered out. After being pooled, the liquidextract was concentrated in a vacuum, and the concentrate was dissolvedin methanol.

The organic layers containing active substance were collected andconcentrated in vacuo. The concentrate was dissolved in methylenechloride and placed on a silica gel (Merck, Art No. 9385) to adsorb theactive substance thereonto. Silica gel column chromatography wasconducted with an ethylacetate-hexane gradient varying from 90:10 to80:20, so as to yield active fractions.

After the adsorption of the fractions onto a C18 column, elution withmethanol and water led to the partial purification of the activesubstance, which was further purified to completion through silica gelcolumn chromatography.

Upon methanol extraction with 2 kg of leaves, about 120 g of the extractwas obtained, from which obovatol and obovatal were yielded in amountsof 25 g and 1.5 g, respectively.

EXAMPLE 2 Structural Analysis of the Purified Compounds

The compounds purified in Example 1 were analyzed for their molecularweights and molecular formulae by analyses including UV absorbance, IR(infrared) absorbance and high resolution mass spectrometry. In detail,a UV-265 spectrophotometer (Shimadzu) was used for UV absorbanceanalysis, a Digilab Division FTS-80 spectrophotometer (Bio-Rad) for IRabsorbance analysis, and high resolution VG70-SEQ mass spectrometry (MS)for the determination of molecular weight and molecular formula. Also,¹H and ¹³C-NMR spectra were obtained using a nuclear magnetic resonator(Varian 300 MHz, 500 MHz NMR) and analyzed to determine the structuresof the compounds.

Physical and chemical properties are given in Table 1 for obovatol andTable 2 for obovatal.

Obovatol

¹H-NMR (CDCl₃): 6.28(H-4, d, J=1.8 Hz), 6.56 (H-6, d, J=1.8 Hz), 3.18(H-7, d, J=6.6 Hz), 5.97 (H-8 and H-8′, m), 5.09 (H-9 and H-9′, m), 6.93(H-2′ and H-6′, d, J=9 Hz), 7.14 (H-3′ and 5′, d, J=9 Hz), 3.36 (H-7′,d, J=6.6 Hz).

Obovatal

¹H-NMR (CDCl₃): 9.35 (H-9, d, J=7.5 Hz), 7.38 (H-7, d, J=15.3 Hz), 7.07(H-3′ and H-5′, d, J=9 Hz), 6.98 (H-4, d, J=1.8 Hz), 6.78 (H-2′ and 6′,d, J=9 Hz), 6.75 (H-6, d, J=1.8 Hz), 6.42 (H-8, dd, J=7.5, 15.3 Hz),4.51 (H-8′, m), 5.09 (H-9′, m), 3.30 (H-7′, d, J=6.6 Hz). TABLE 1Appearance pale green Empirical Molecular C₁₈H₁₈O₃ Formula Mw 282 m.p.(°C.) Liquid Solubility Soluble Alcohol, DMSO Insoluble Hexane, H₂O

TABLE 2 Appearance Pale yellow Empirical Molecular C₁₈H₁₆O₃ Formula Mw280 m.p.(° C.) 161-162° C. Solubility Soluble Alcohol, DMSO InsolubleH₂O

EXAMPLE 3 Inhibition of Growth of Cancer Cell Strain

The compounds isolated and purified in Example 1 were assayed for theirability to inhibit the growth of cancer cell strains using WST-1. Humancancer cell strains were cultured in media supplemented with 10% FBS inan incubator at 37° C. under 5% CO₂ atmosphere, and cell cultures weresplit when they reached confluence, using 0.05% trypsin-EDTA.

After the determination of cell number using a hematocytometer, cellswere placed in each well of 96-well plates at densities of 4,000 cells(A549, MDA-MB-231), 5,000 cells (HEK293, NCI-H23) and 6,000 cells(HCA-7, HCT116, SW620, DU145).

24 hours after incubating the cells in a medium supplemented with 10%FBS in a 37° C. 5% CO₂ incubator, the medium was changed with freshmedium containing a control (0.1% DMSO) or various concentrations of thecompounds purified in Example 1 (solutions of the compounds in DMSO werediluted with the medium). After incubation for 48 hours, the addition of10 μl of WST-1 (Roche) to each well was followed by incubation for 2hours. Absorbance at 450 mm was measured using an ELISA reader(Bio-Rad).

ELISA analysis indicated that when used in an amount from 5 to 15 μg/ml,the compounds of the present invention inhibited the growth of cancercells by 50%, and were effective for different cancer cell strains.Obovatol was found to have the most inhibitory activity against humanlarge intestine cancer cells HCT116 and HCA-7, as indicated by measuredGI₅₀ values of 5 μg/ml and 6 μg/ml respectively. On the other hand,obovatol was measured to have a GI₅₀ value of 10 μg/ml against SW620 (acolon carcinoma), 20 μg/ml and 25 μg/ml against NCI-H23 and A549 (bothlung cancer), respectively, 18 μg/ml against DU145 (prostate cancer), 31μg/ml against MDA-MB-231 (breast cancer), and 25 μg/ml against HEK293(kidney cancer). As for obovatal, its GI₅₀ values were measured to be 7,10 and 13 μg/ml against HCT116, HCA-7, SW620 (all colon carcinomacells), respectively, 11 and 17 μg/ml against NCI-H23 and A549 (bothlung cancer), respectively, 20 μg/ml against MDA-MB-231 (breast cancer),and 21 μg/ml against HEK293 (kidney cancer).

EXAMPLE 4 Determination of Ability of Obovatol and Obovatal to InhibitExpression of MMP9

The human fibrosarcoma HT1080 was incubated overnight at a density of1×10⁵ cells/ml in each well of 96-well plates containing a mediumsupplemented with 10% FBS, followed by exchange of the medium with afresh serum-free medium. Three hours after treatment with samples, thecells were treated with 5 ng/ml of tumor necrosis factor α (TNF-α) andthen incubated for 17 hours.

Observations were made of cell morphology and cytotoxicity. Onlysupernatants of the cell cultures were mixed with a storage buffercontaining glycerol and a chromogenic reagent and subjected toelectrophoresis, which indicated that MMP-9 expression was greatlyinhibited in the treated cells compared to the control. TNF-α, acting asan inducer to activate cancer cells in the control, was monitored withthe naked eye for its induction behaviors in treated and non-treatedcells. In the culture media treated with the samples, a reduced level ofMMP-9 was detected as measured by SDS-PAGE.

According to data, obovatol and obovatal inhibited the expression ofMMP9 by 50% or more at concentrations of 10 and 5 μg/ml, respectively.

EXAMPLE 5 Determination of Ability of Obovatol and Obovatal to InhibitExpression of MMP2

The pro-enzyme MMP2 was activated with 0.5 mM p-aminophenylmercuricacetate (APMA) in a reaction buffer at 37° C. for 15 min, and thenmeasured for reaction rate in TNBC buffer (20 mM Tris-HCl, 5 mM CaCl₂,0.15 M NaCl, pH 7.5), with7-methoxycoumarin-4-yl-acetyl-Pro-Leu-Gly-Leu-(2-[2,4-dinitrophenyl]-2,3-diaminopropionyl)-Ala-Arg-NH₂(Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂) serving as a substrate.

While reacting with the substrate, samples treated with obovatol andobovatal were measured at regular intervals for absorbance at 480 nm.Comparison with control showed that obovatol and obovatal inhibited theactivity of MMP2 by 50% or more at concentrations of 0.3 and 90 μg/ml,respectively.

EXAMPLE 6 Acute Oral Toxicity Assay in Rats

Using six-week-old specific pathogen-free (SPF) SD rats, an acutetoxicity assay was conducted. The rats were divided into groups, eachconsisting of two rats. After being dissolved in injectable saline, theobovatol or obovatal obtained in Example 1, was orally administered oncein a dosage of 1 g/kg/ml to the rat groups. Afterwards, observationswere made of the death, clinical symptoms, and weight changes of theanimals, and serological and serobiochemical assays were conducted.Also, an autopsy was carried out to examine abnormalities of abdominaland thoracic organs with the naked eye.

None of the animals to which the compounds of interest were administeredexhibited noticeable clinical symptoms or died. Cytotoxicity was notobserved in the weight change, serological assay, serobiochemical assay,or autopsy observations for the animals administered with the compoundsof the present invention.

Causing no toxic effects to a dose of 500 mg/kg, both obovatol andobovatal, purified according to the present invention, were determinedto have an oral lethal dose (LD₅₀) of 500 mg/kg and thus be regarded assafe.

INDUSTRIAL APPLICABILITY

As described hereinbefore, obovatol, represented by Chemical Formula 1,and obovatal, represented by Chemical Formula 2, exhibit activity ofinhibiting the growth of cancer cells and suppressing the expression andactivity of matrix metalloproteinases (MMPs), and thus can be useful forthe prophylaxis and treatment of cancer as well as for the inhibition ofcancer metastasis.

Although the preferred embodiments of the present invention have beendisclosed for illustrative purposes, those skilled in the art willappreciate that various modifications, additions and substitutions arepossible, without departing from the scope and spirit of the inventionas disclosed in the accompanying claims.

1. An anticancer composition, comprising obovatol, represented byChemical Formula 1, obovatal, represented by Chemical Formula 2, and/orpharmaceutical salts thereof.


2. The anticancer composition as set forth in claim 1, wherein theanticancer composition is effective to treat cancer selected from amonglarge intestine cancer, rectal cancer, stomach cancer, prostate cancer,breast cancer, kidney cancer, liver cancer, encephaloma, lung cancer,uterine cancer, colon cancer, bladder cancer, pancreatic cancer, andblood cancer.
 3. A method for the prophylaxis or treatment of cancer,comprising the administration of an anticancer composition comprisingobovatol, represented by the following Chemical Formula 1, obovatal,represented by the following Chemical Formula 2, and/or pharmaceuticalsalts thereof.


4. A method for inhibiting the metastasis of cancer, comprising theadministration of an anticancer composition comprising obovatol,represented by the following Chemical Formula 1, obovatal, representedby the following Chemical Formula 2, and/or pharmaceutical saltsthereof.